bioRxiv preprint doi: https://doi.org/10.1101/854851; this version posted December 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 2 Functions of Gtf2i and Gtf2ird1 in the developing brain: transcription, DNA-binding, 3 and long term behavioral consequences. 4 5 6 Authors: 7 Nathan D. Kopp1,2, Kayla R. Nygaard1,2, Katherine B. McCullough1,2, Susan E. Maloney2,3, 8 Harrison W. Gabel4, Joseph D. Dougherty1,2,3,* 9 10 11 Affiliations: 12 13 1Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA 14 2Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA 15 3Intellectual and Developmental Disabilities Research Center, Washington University School 16 of Medicine, St. Louis, MO, USA 17 4Department of Neuroscience, Washington University School of Medicine, St Louis MO, USA 18 19 Contact Information: 20 Dr. Joseph Dougherty 21 Department of Genetics 22 Campus Box 8232 23 4566 Scott Ave. 24 St. Louis, MO. 63110-1093 25 P: 314-286-0752 26 F: 314-362-7855 27 E: [email protected] 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 1 bioRxiv preprint doi: https://doi.org/10.1101/854851; this version posted December 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 2 Abstract 3 4 Gtf2ird1 and Gtf2i may mediate aspects of the cognitive and behavioral phenotypes 5 of Williams Syndrome (WS) – a microdeletion syndrome encompassing these 6 transcription factors (TFs). Knockout mouse models of each TF show behavioral 7 phenotypes. Here we identify their genomic binding sites in the developing brain, 8 and test for additive effects of their mutation on transcription and behavior. Both 9 TFs target constrained chromatin modifier and synaptic protein genes, including a 10 significant number of ASD genes. They bind promoters, strongly overlap CTCF 11 binding and TAD boundaries, and moderately overlap each other, suggesting 12 epistatic effects. We used single and double mutants to test whether mutating both 13 TFs will modify transcriptional and behavioral phenotypes of single Gtf2ird1 14 mutants. Despite little difference in DNA-binding and transcriptome-wide 15 expression, Gtf2ird1 mutation caused balance, marble burying, and conditioned fear 16 phenotypes. However, mutating Gtf2i in addition to Gtf2ird1 did not further modify 17 transcriptomic or most behavioral phenotypes, suggesting Gtf2ird1 mutation alone 18 is sufficient. 19 20 21 22 23 24 25 2 bioRxiv preprint doi: https://doi.org/10.1101/854851; this version posted December 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 2 Introduction 3 4 The Williams syndrome critical region (WSCR) contains 28 genes that are 5 typically deleted in Williams syndrome (WS) (OMIM#194050). The genes in this 6 region are of interest for their potential to contribute to the unique physical, 7 cognitive, and behavioral phenotypes of WS, which include craniofacial 8 dysmorphology, mild to severe intellectual disability, poor visuospatial cognition, 9 balance and coordination problems, and a characteristic hypersocial personality (1– 10 3). Single gene knockout mouse models exist for many of the genes in the region, 11 with differing degrees of face validity for WS phenotypes (4–9). Two genes have 12 been highlighted in the human and mouse literature as playing a large role in the 13 social and cognitive tasks: Gtf2i and Gtf2ird1. Mouse models of each gene have 14 shown social phenotypes as well as balance and anxiety phenotypes (4,8–12). Since 15 evidence shows that each gene affects similar behaviors, we set out to test the 16 hypothesis that simultaneous knockdown of both genes would cause more severe 17 phenotypes. Investigating the genes together, rather than individually, could 18 provide a more complete understanding of how the genes in the WSCR contribute to 19 the phenotypes of WS. 20 Gtf2i and Gtf2ird1 are part of the General Transcription Factor 2i family of 21 genes. A third member, Gtf2ird2, is located in the WSCR that is variably deleted in 22 WS patients with larger deletions (13). This gene family arose from gene duplication 23 events, resulting in high sequence homology between the genes (14). The defining 3 bioRxiv preprint doi: https://doi.org/10.1101/854851; this version posted December 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 feature of this gene family is the presence of the helix-loop-helix I repeats, which are 2 involved in DNA and protein binding (15). Gtf2i’s roles include regulating 3 transcriptional activity in the nucleus. However, this multifunctional transcription 4 factor also exists in the cytoplasm where it conveys messages from extracellular 5 stimuli and regulates calcium entry into the cell (16,17). So far, Gtf2ird1 has only 6 been described in the nucleus of cells and is thought to regulate transcription and 7 associate with chromatin modifiers (18). The DNA binding of these two 8 transcription factors (TFs) has only been studied in ES cells and embryonic 9 craniofacial tissue. They recognize similar and disparate genomic loci, suggesting 10 the proteins interact to regulate specific regions of the genome (19,20). However, 11 binding of these TFs has not been studied in the developing brain, which could 12 provide more relevant insight on how the General Transcription Factor 2i family 13 contributes to cognitive and behavioral phenotypes. 14 We performed ChIP-seq on GTF2I and GTF2IRD1 in the developing mouse 15 brain to define where these TFs bind and then tested the downstream consequences 16 of disrupting this binding. We used the CRISPR/Cas9 system to make a mouse model 17 with a mutation in Gtf2ird1 alone and a mouse model with mutations in both Gtf2i 18 and Gtf2ird1 to test how adding a Gtf2i mutation modifies the effects of Gtf2ird1 19 mutation. We showed the mutation in Gtf2ird1 resulted in the production of an N- 20 truncated protein that disrupts the binding of GTF2IRD1 at the Gtf2ird1 promoter 21 and deregulates the transcription of Gtf2ird1, moderately decreasing protein levels 22 so homozygous mutants approximate levels expected from hemizygosity of the 23 WSCR. While there are mild consequences of the mutation on genome-wide 4 bioRxiv preprint doi: https://doi.org/10.1101/854851; this version posted December 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 transcription, the mutant mouse exhibited clear balance, marble burying, and 2 conditioned fear differences. Comparing the single gene mutant to the double 3 mutant did not reveal more severe transcriptional changes and behavioral 4 phenotypes, however adding Gtf2i on top of a background of two Gtf2ird1 mutations 5 resulted in abnormal pre-pulse inhibition and cued fear conditioning. This suggests 6 Gtf2ird1 drives the majority of the phenotypes observed in current studies, and total 7 protein level, N-terminal truncation, or both have functional consequences on DNA- 8 binding and behavior. 9 10 Results 11 12 Gtf2i and Gtf2ird1 bind at active promoters and conserved sites 13 14 The paralogous transcription factors, GTF2I and GTF2IRD1, have been 15 implicated in the behavioral phenotypes seen in humans with WS as well as mouse 16 models (4,8,11,12,21,22). However, the underlying mechanisms by which the 17 General Transcription Factor 2i family acts are not well understood. One approach 18 to begin to identify how these TFs can regulate phenotypes is by identifying where 19 they bind in the genome. This has been done in ES cells and embryonic facial tissue 20 and revealed that both of these transcription factors bind to genes involved in 21 craniofacial development (19). However, these are not relevant tissues when 22 considering phenotypes related to brain development and subsequent behavior. To 23 address this, we performed ChIP-seq for GTF2IRD1 and GTF2I in the developing 5 bioRxiv preprint doi: https://doi.org/10.1101/854851; this version posted December 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 brain at embryonic day 13.5 (E13.5), a time point when both of these proteins are 2 highly expressed (23). 3 We identified 1,410 peaks that were enriched in the GTF2IRD1 IP samples 4 compared to the input (Supplemental Table 1). The GTF2IRD1-bound regions 5 were strikingly enriched in the promoters of genes and along the gene body, more 6 so than would be expected by randomly sampling the genome (Figure 1A) (χ2 = 7 1537.8, d.f. =7, p < 2.2x10-16). The bound peaks were found mostly in H3K4me3 8 bound regions (OR=779.5, p<2.2x10-16 Fisher’s exact test (FET)), suggesting they are 9 in active sites in the genome.